The long term objective of this project is to understand the signaling pathways which regulate the activity of heme oxygenase-2 (HO2), which catalyzes the conversion of carbon monoxide (CO), biliverdin. and iron. CO formed by the action of H02 has been proposed to function as a neurotransmitter. Furthermore, bilirubin (rapidly formed from biliverdin by the enzyme biliverdin reductase) has been shown to be a potent antioxidant which confers neuroprotection against oxidative damage. However, the role of CO as a neurotransmitter remains controversial because H02 has not been shown to be activated by a signal transduction pathway. The specific aims of this project are as follows: 1) We will determine the functional effects of various protein kinases on the enzymatic activity of H02 and determine the molecular mechanisms by which H02 is activated in vivo by signal transduction pathways. 2) We will develop a novel assay to measure H02 activity in real time in intact cells by exploiting recently developed iron-sensitive dyes. Once developed this assay will then be used to monitor H02 activity in neuronal cultures in response to multiple stimuli including membrane depolarization and agonist stimulation. 3) We will elucidate the signaling pathways responsible for the activation of H02 response to oxidative damage. This will be accomplished by the combination of the use of phospho-specific H02 antibodies, phannacological agents, and real-time monitoring of H02 activity in primary neuronal cultures.